The ryanodine receptor type 1 (RyR1) plays a vital role in skeletal muscle contraction by releasing Ca2+ required for excitation-contraction (EC) coupling. A key structural domain in RyR1 called the clamp region mediates activation of RyR1 during muscle contraction. Missense mutations in the clamp region can cause debilitating skeletal muscle disorders including malignant hyperthermia (MH) and central core disease (CCD). However, the overall structure of this critical regulatory domain and the effects of these mutations on conformational changes that occur during RyR1 activation are largely unknown. The objective of this proposal is to utilize an innovative site-specific labeling method combined with Forster resonance energy transfer (FRET) techniques to examine conformational changes that occur in the clamp region of RyR1 expressed in intact cells. The long-term goal of the proposed work is to compare conformational changes occurring in wildtype and MH/CCD mutated RyR1 to determine how structural rearrangements of RyR1 caused by these mutations can lead to these skeletal muscle disorders. Hypothesis: RyR1 activation involves changes in domain-domain interactions within the clamp region that are disrupted by point mutations that lead to skeletal muscle disorders. Specific Aims: In Aim 1, a FRET-based assay will be used to measure conformational changes between 3 putative sub-domains found in the first 600 amino acids of RyR1. A FRET pair consisting of a donor, green fluorescent protein (GFP) fused into RyR1 and a fluorescent acceptor synthesized by the P.I. (Cy3NTA) targeted to 10 residue histidine (His10) tags inserted into the RyR1 primary sequence will be used for FRET measurements in intact cells and in vitro. Changes in FRET after alteration of channel activity by physiological (i.e. EC coupling) and pharmacological modulators will indicate conformational changes occurring between the donor and acceptor sites. Changes in these conformational movements resulting from introduction of MH and CCD mutations will also be determined. In Aim 2, this FRET assay will be used to examine a specific structural domain switch hypothesis that suggests that MH is caused by a defective interaction between two RyR1 domains (MH zone 1 and 2). Measurements of changes in FRET from GFP in MH Zone 1 to His10 tags inserted into MH zone 2 that occur during EC coupling or as the result of bioactive domain peptides or MH/CCD mutations will be used to probe this putative interaction between these two domains. Perspective: Through this integrated series of experiments, a new set of molecular tools developed to site-specifically label RyR1 will be used to examine dynamic changes in protein structure involved in channel gating. These experiments should provide unprecedented glimpses of the structure of RyR1 and how this structure is perturbed by disease-causing mutations.